The Antiproliferative Effect And Mechanism Of Cardamonin On MTOR Inhibitor Resistant Tumor Cells

Aim1 To confirm the antiproliferative effect of cardamonin on mTOR inhibitors?rapamycin or AZD8055?resistant HeLa cells and MCF-7 cells.2 To explore the mechanism of the antiproliferative effect of cardamonin on rapamycin?RAP?or AZD8055?AZD?resistant cells.Methods1 The resistant clones of HeLa cells and MCF-7 cells were constructed by increasing concentrations of either rapamycin or AZD8055HeLa cells and MCF-7 cells were exposed to RAP or AZD by increasing concentrations to induce and construct resistant clones?HeLa/RAP,MCF-7/RAP,HeLa/AZD and MCF-7/AZD?.The degree of resistance for RAP or AZD was identified by cytotoxicity and Western blot.2 Clones from single cell source were harvested by limited dilution method3 Cell cultureCells of HeLa,HeLa/RAP and HeLa/AZD were cultured in 1640 medium,and cells of MCF-7,MCF-7/RAP and MCF-7/AZD were cultured in High-glucose DMEM medium under the conventional condition(37°C,5%CO₂ and medium including 10%fetal bovine serum,100?g·mL^-1 streptomycin and 100 U·mL^-1penicillin).Cells were adherently grown with single-layer to 80%⁹0%and then digested by 2.5%trypsin and passaged.4 Groups and drugs intervention4.1 Cell groups:Cells were divided into 6 groups?HeLa,MCF-7,HeLa/RAP,MCF-7/RAP,HeLa/AZD and MCF-7/AZD?.4.2 Drugs intervention:Normal control group,RAP groups?30,300,3000 nM?,AZD groups?30?300?3000 nM?and cardamonin?CAR?groups?10,30?M?were set in each cell groups to detect the cell clone formation and cell migration;normal control group,RAP groups?3,30,300 nM?,AZD groups?3,30,300 nM?and CAR group?10,30?M?were set to detect the expression of related proteins.5 Measurements and methodology5.1 Cell morphology and cell growth were observed by an inverted microscope.5.2 Cell cytotoxicity and cell growth curve were measured by MTT method with RAP,AZD and CAR treatment.5.3 Cell clone formation was tested by tablet cloning experiment with RAP,AZD and CAR treatment.5.4 Cell migration was evaluated by Scratch test with RAP,AZD and CAR treatment.5.5 Western blot was used to detect the expression of multi-drug resistance protein 1 ?MDR1?,?-catenin,key proteins of mTOR signaling pathway?AKT,p-AKT,S6K1,p-S6K1,4E-BP1,p-4E-BP1,mTOR and p-mTOR?and its binding protein ?Raptor?.Results1 RAP resistant clones?HeLa/RAP,MCF-7/RAP?and AZD resistant clones?HeLa/AZD,MCF-7/AZD?were constructed successfully.The resistance index of HeLa/RAP,HeLa/AZD,MCF-7/RAP and MCF-7/AZD was 5.89,24.90,68.24 and21.23,respectively.The ability of cell proliferation and cell clone formation was consistently decreased in drug-resistant cells.2 CAR had antiproliferative effect on RAP or AZD resistant cells.There was no difference in the half inhibitory concentration of CAR on non-resistant cells and drug-resistant cells.However,clone formation was significantly inhibited by CAR on RAP or AZD resistant cells?P<0.01?.3 Cell migration were significantly inhibited by CAR on RAP or AZD resistant cells?P<0.01?.High expressions of?-catenin were observed on drug-resistant cells.CAR inhibited the expression of?-catenin in a concentration-dependent way?P<0.01?.4 The expression of p-mTOR,p-S6K1 and the total protein of Raptor were significantly inhibited by CAR in RAP or AZD resistant cells?P<0.01?.5 Expression of MDR1 was higher on drug-resistant cells.CAR inhibited the expression?P<0.01?.Conclusions1 Cell proliferation and cell migration were suppressed by CAR in RAP or AZD resistant cells?HeLa/RAP,MCF-7/RAP,HeLa/AZD and MCF-7/AZD?.2 The mechanism of resistance of HeLa and MCF-7 cells to RAP and AZD may be related to the high expression of MDR1 protein.3 The possible mechanisms of CAR on antiproliferative effect were involved in inhibition of the expression of mTORC1 specific binding protein?Raptor?and phosphorylation levels of mTORC1 signaling pathway proteins.In addition,it was also closely related to suppressing the protein expression of?-catenin and MDR1 in RAP or AZD resistant cells.